Molecular Epidemiology Of Mycobacterium At The Animal Human Interface And Its Co-Morbidity With Diabetes Mellitus

Material type: BookPublisher: 2016Description: 153p.Subject(s): Epidemiology and Public Health | Phd. ThesesDDC classification: 2710-T Dissertation note: Tuberculosis (TB) is a common and fatal infectious disease which has afflicted mankind for several millennia. At the moment, TB is positioned at number five when it comes to the most common causes of fatality worldwide. TB is curable if it is properly diagnosed and treated. In 2015, it was estimated that 1.5 million deaths (an equivalent of 4,000 deaths per day) and 9 million new TB cases have been reported. Diabetes Mellitus is also widely distributed and estimated to affect 366 million people by 2030. The co-morbidity of DM and TB is re-emerging because of the progressive epidemiology of both diseases especially in the developing countries. Endemicity of TB and DM is growing in developing countries because of low socio-economic status and poor living conditions.
In this study, a total of 500 tuberculosis positive patients were selected under TB DOTS program from five tertiary care hospitals of Lahore. Sputum samples were collected from all the enrolled patients and smear microscopy was performed for TB confirmation. Blood samples were collected from the same patients for screening of diabetes mellitus. Sputum samples were also processed for culture and drug sensitivity on LJ medium. Molecular identification by PCR technique was carried out on all positive cultured strains and results were compared with reference strain H37RV. For DNA sequencing, PCR products were sent to Singapore where sequencing was performed by Sanger method.
Data was compiled and variables including gender, age, drug resistance and treatment history and correlation among different variables was analyzed using chi-square test and Fischer’s exact test method at P-value of ≤0.05. SPSS (Statistical Package for Social Sciences, Version 20.0) was used for statistical analysis. The count data was statistically analyzed using
SUMMARY
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descriptive statistical tools. On screening for fasting blood sugar level, 74 (14.8%) patients were recorded as diabetics as well i.e. blood sugar level ≥ 126 mg/dl. Out of these 74 patients, 22 patients had previous history of diabetes whereas remaining 52 patients were newly diagnosed at the time of screening. The maximum distribution of TB-DM patients was found in age group > 57 years. Mean age of the group without DM was 39 years and with DM was 48 years. Coexistence of DM in TB patients was higher in males (62.2%) as compared to female study subjects. However, the gender difference is statistically non-significant (p value 0.243).
The distribution of education level revealed that out of the total participants, maximum number of patients (n=220) were illiterate and similar trend was observed in diabetic patients with 54 (73%) individuals belonging to the illiterate group of the subjects. There is statistically significant difference between existence of DM and literacy level in tuberculosis patients. Among social and behavioral risk factors in tuberculosis patients, majority of the patients were unemployed (24%) in TB-DM group. Significant correlation p value ≤ 0.05 was found between coexistence of TB-DM and tobacco use. TB cases with diabetes were known to have history of smoking with 73% (n=54) while non-smokers were 27% (n=20).
On sputum smear microscopy frequency of 3+ results showing high bacterial load, was profoundly higher i.e. 67.6% in diabetic tuberculosis patients as compared to non-diabetics which was 4.9% only. Total culture yield was 363 out of 500 sputum samples. There were 193 samples that were sensitive to all drugs, 9.4% were MDR strains (resistant to Isoniazid and Rifampicin). MDR-TB is significantly higher in TB-DM patients i.e. 13.5% as compared to 8.7% in TB only patients.
In our study, DNA sequence data for drug resistance was studied by the sequence of rpoB gene of the wild type MTB strain. Sequencing results showed mutations at various spots of rpoB gene.
SUMMARY
125
Most common mutational sites identified were at codon 531, 526 and 516 with frequency of 70%, 15% and 7.5%, respectively. Moreover, mutation sites at 512 and 574 codon had also been reported. In this study, predominantly two phylogenetic variants were identified. Majority of the isolated strains were Central Asia Strain (CAS) with a prevalence of 88.2% and rest were Beijing strain. However, attempts to find zoonosis could not be established. A total of 900 raw milk samples were also screened for M. bovis and no positive sample could be detected.
The present study emphasizes the importance of screening for DM in TB patients, which had not been done in routine. This practice may prove to be helpful in reducing the disease burden of TB patients as well as DM patients. Thus it is recommended that the screening for DM should be implemented in TB/DOTS clinics.
Emergence of Multi drug resistant Mycobacterium tuberculosis is also a serious challenge for clinicians. A very large financial implication in terms of treatment, duration of chemotherapy and spread of MDR TB strains is being faced. Treating MDR TB is more complicated than treating drug sensitive TB. Patients with MDR TB require longer, much more costly treatment and experience higher mortality rates. Such a long time to initiate the treatment is not affordable, thus there is a dire need for some rapid technique like molecular based diagnostics for MDR detection, which can provide quick results and making it possible to start treatment at earlier to minimize transmission, morbidity and mortality.

Tuberculosis (TB) is a common and fatal infectious disease which has afflicted mankind for several millennia. At the moment, TB is positioned at number five when it comes to the most common causes of fatality worldwide. TB is curable if it is properly diagnosed and treated. In 2015, it was estimated that 1.5 million deaths (an equivalent of 4,000 deaths per day) and 9 million new TB cases have been reported. Diabetes Mellitus is also widely distributed and estimated to affect 366 million people by 2030. The co-morbidity of DM and TB is re-emerging because of the progressive epidemiology of both diseases especially in the developing countries. Endemicity of TB and DM is growing in developing countries because of low socio-economic status and poor living conditions.
In this study, a total of 500 tuberculosis positive patients were selected under TB DOTS program from five tertiary care hospitals of Lahore. Sputum samples were collected from all the enrolled patients and smear microscopy was performed for TB confirmation. Blood samples were collected from the same patients for screening of diabetes mellitus. Sputum samples were also processed for culture and drug sensitivity on LJ medium. Molecular identification by PCR technique was carried out on all positive cultured strains and results were compared with reference strain H37RV. For DNA sequencing, PCR products were sent to Singapore where sequencing was performed by Sanger method.
Data was compiled and variables including gender, age, drug resistance and treatment history and correlation among different variables was analyzed using chi-square test and Fischer’s exact test method at P-value of ≤0.05. SPSS (Statistical Package for Social Sciences, Version 20.0) was used for statistical analysis. The count data was statistically analyzed using
SUMMARY
124
descriptive statistical tools. On screening for fasting blood sugar level, 74 (14.8%) patients were recorded as diabetics as well i.e. blood sugar level ≥ 126 mg/dl. Out of these 74 patients, 22 patients had previous history of diabetes whereas remaining 52 patients were newly diagnosed at the time of screening. The maximum distribution of TB-DM patients was found in age group > 57 years. Mean age of the group without DM was 39 years and with DM was 48 years. Coexistence of DM in TB patients was higher in males (62.2%) as compared to female study subjects. However, the gender difference is statistically non-significant (p value 0.243).
The distribution of education level revealed that out of the total participants, maximum number of patients (n=220) were illiterate and similar trend was observed in diabetic patients with 54 (73%) individuals belonging to the illiterate group of the subjects. There is statistically significant difference between existence of DM and literacy level in tuberculosis patients. Among social and behavioral risk factors in tuberculosis patients, majority of the patients were unemployed (24%) in TB-DM group. Significant correlation p value ≤ 0.05 was found between coexistence of TB-DM and tobacco use. TB cases with diabetes were known to have history of smoking with 73% (n=54) while non-smokers were 27% (n=20).
On sputum smear microscopy frequency of 3+ results showing high bacterial load, was profoundly higher i.e. 67.6% in diabetic tuberculosis patients as compared to non-diabetics which was 4.9% only. Total culture yield was 363 out of 500 sputum samples. There were 193 samples that were sensitive to all drugs, 9.4% were MDR strains (resistant to Isoniazid and Rifampicin). MDR-TB is significantly higher in TB-DM patients i.e. 13.5% as compared to 8.7% in TB only patients.
In our study, DNA sequence data for drug resistance was studied by the sequence of rpoB gene of the wild type MTB strain. Sequencing results showed mutations at various spots of rpoB gene.
SUMMARY
125
Most common mutational sites identified were at codon 531, 526 and 516 with frequency of 70%, 15% and 7.5%, respectively. Moreover, mutation sites at 512 and 574 codon had also been reported. In this study, predominantly two phylogenetic variants were identified. Majority of the isolated strains were Central Asia Strain (CAS) with a prevalence of 88.2% and rest were Beijing strain. However, attempts to find zoonosis could not be established. A total of 900 raw milk samples were also screened for M. bovis and no positive sample could be detected.
The present study emphasizes the importance of screening for DM in TB patients, which had not been done in routine. This practice may prove to be helpful in reducing the disease burden of TB patients as well as DM patients. Thus it is recommended that the screening for DM should be implemented in TB/DOTS clinics.
Emergence of Multi drug resistant Mycobacterium tuberculosis is also a serious challenge for clinicians. A very large financial implication in terms of treatment, duration of chemotherapy and spread of MDR TB strains is being faced. Treating MDR TB is more complicated than treating drug sensitive TB. Patients with MDR TB require longer, much more costly treatment and experience higher mortality rates. Such a long time to initiate the treatment is not affordable, thus there is a dire need for some rapid technique like molecular based diagnostics for MDR detection, which can provide quick results and making it possible to start treatment at earlier to minimize transmission, morbidity and mortality.

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